Method of testing anticancer agent-sensitivity of tumor cells

ABSTRACT

Susceptibility of a tumor cell to an anticancer agent is tested by measuring an expression level or levels of a gene or genes selected from the group consisting of the genes listed in Tables 3 and 4 in a tumor cell collected from a cancer patient treated with an anticancer agent (E7070 and related compounds thereof), or causing the anticancer agent to act on a tumor cell collected from a cancer patient and measuring an expression level or levels of a gene or genes selected from the group consisting of the genes listed in Tables 3 and 4 in the tumor cell, and determining that the tumor cell is susceptible to the anticancer agent when the expression level or levels of the gene or genes listed in Table 3 increase, or the expression level or levels of the gene or genes listed in Table 4 decrease.

This is the U.S. National Phase under 35 U.S.C. § 371 of InternationalApplication PCT/JP01/10282, filed Nov. 26, 2001, which was published ina language other than English which claims priority of JP 2000-357398,filed Nov. 24, 2000. Each of the above applications are incorporatedherein by reference in their entirety.

TECHNICAL FIELD

The present invention relates to a method for testing whether a tumorcell is susceptible to an anticancer agent or not.

BACKGROUND ART

In a conventional clinical study of an anticancer agent, its toxicityprofile and maximum recommended dose are first determined in the Phase Iclinical study, and then the agent is evaluated as a drug based on theresponse rate using the tumor reduction rate as a criterion for efficacyin the Phase II clinical study. Meanwhile, with the advancement incancer biology in recent years, drugs having a novel action mechanismfor inhibiting the intracellular signal transduction system,angiogenesis and so forth are in the course of active research anddevelopment. For these novel anticancer agents, it may be possible thata maximum recommended dose close to a toxic dose does not necessarilyneed to be administered. Further, it is estimated that drug efficacycould be more appropriately judged by using improvement of QOL (Qualityof Life) or prolongation of life associated with tumor growth inhibitionas an index rather than tumor reduction. In this case, to more logicallyand specifically confirm the drug efficacy, it is desirable to utilizechange of a biological marker closely relating to a tumor growthinhibition mechanism as a surrogate marker.

In the anticancer therapy, in general, reactivity of a living body whenan anticancer agent is administered is largely depends on susceptibilityof a tumor cell, which is a target of the drug, to the drug. Generally,this susceptibility of tumor cell to the drug greatly varies in everytype of tumor cell. Such differences in susceptibility are attributableto quantitative or qualitative differences of target molecules of thedrug or factors relating to the molecules, acquisition of drugresistance and so forth. Considering such a background, it would be veryuseful if change in a tumor cell specifically caused when the tumor cellas a target exhibits susceptibility to a drug can be measured by usingtumor tissue obtained by biopsy etc., because early determination ofdrug efficacy, establishment of a treatment method, selection of a newtreatment method and so forth become possible by using the change as asurrogate marker. Further, if a tumor cell is isolated from tumor tissueobtained by biopsy or the like in a conventional manner prior to atreatment and then treated with a drug, and whether this tumor cell issusceptible to the drug is determined based on change of theaforementioned surrogate marker, it becomes possible to preliminarilypredict whether the treatment using the drug is effective or not, andthis would be extremely useful in clinical practice. It is importantthat the change of this surrogate marker should be specific to antitumoreffect, and it is sufficient that the change can be measured with highsensitivity. Specifically, quantification of variations in the geneexpression specific to the antitumor effect of the drug, analysis ofquantitative variations of a protein along with the changes in geneexpression, analysis of functional changes associated with these changesand so forth can be used as the surrogate markers.

E7070 (N-(3-chloro-7-indolyl)-1,4-benzenedisulfonamide) is a compoundthat has an antitumor effect targeting the G1 phase of the cell cycle,and is being clinically developed (Takashi Owa, Hiroshi Yoshino, TatsuoOkauchi, Kentaro Yoshimatsu, Yoichi Ozawa, Naoko Hata Sugi, TakeshiNagasu, Nozomu Koyanagi and Kyosuke Kitoh, J. Med. Chem, 1999, 42,3789-3799).

Spectra of intensity of the growth inhibitory action of this compound onvarious tumor cells are different from those of any of existinganticancer agents, and this compound is expected to have an effect as ananticancer agent having a novel action mechanism. In order to acceleratethe clinical development of this drug and early establish a clinicaltreatment method, and contribute to improvement of QOL of patients byefficiently advancing treatment based on established treatment methods,it is desirable to discover and apply a surrogate marker that can bespecifically used upon the administration of the drug.

In recent years, methods of using various DNA microarrays to detectexpression levels of a large number of genes at the same time have beenestablished and widely used (Schena M, Shalon D, Davis R W, Brown P O,Science 1995, 270, 467-70; Lockhart, D. J., Dong, H., Byrne, M. C.,Follettie, M. T., Gallo, M. V., Chee, M. S., Mittmann, M., Wang C.,Kobayashi, M., Horton, H. Brown, E. L., Nature Biotechnology, 1996, 14,1675-1680).

Also in the field of cancer studies, researches using such DNAmicroarrays are actively being conducted. For example, in a study inwhich diffuse large B-cell lymphoma (DLBCL) was investigated byexpression analysis using a DNA microarray, DLBCL has been classifiedinto two of different types according to differences in gene expressionprofiles, and it has been shown that this classification leads toprediction of prognosis (Alizadeh A A, Eisen M B, Davis R E, Ma C,Lossos I S, Rosenwald A, Boldrick J C, Sabet H, Tran T, Yu X, Powell JI, Yang L, Marti G E, Moore T, Hudson J Jr, Lu L, Lewis D B, TibshiraniR, Sherlock G, Chan W C, Greiner T C, Weisenburger D D, Armitage J O,Warnke R, Staudt L M, et al, Nature, 2000, 403, 503-11). Further, therehave been a report in which, by analyzing gene expression profiles of apanel of 60 types of cancer cell lines from the National CancerInstitute in the United States, these cell lines were reclassified andtheir characteristics were examined (Ross D T, Scherf U, Eisen M B,Perou C M, Rees C, Spellman P, Iyer V, Jeffrey S S, Van de Rijn M,Waltham M, Pergamenschikov A, Lee J C, Lashkari D, Shalon D, Myers T G,Weinstein J N, Botstein D, Brown P O, Nat Genet, 2000, 24, 227-35), areport in which relationships of the gene expression profiles of thepanel of 60 types of cancer cell lines and susceptibility to variousanticancer agents of each cell line were discussed (Scherf U, Ross D T,Waltham M, Smith L H, Lee J K, Tanabe L, Kohn K W, Reinhold W C, Myers TG, Andrews D T, Scudiero D A, Eisen M B, Sausville E A, Pommier Y,Botstein D, Brown P O, Weinstein J N, Nat Genet, 2000, 24, 236-44) andso forth.

Further, there have also been several reports in which changes in geneexpression that occurred when anticancer agents were caused to act ontumor cells were examined by similarly using a DNA microarray (partlymacroarray using a membrane filter) (Rhee C H, Ruan S, Chen S, ChenchikA, Levin V A, Yung A W, Fuller G N, Zhang W, Oncol Rep, 1999, 6,393-401. Zimmermann J, Erdmann D, Lalande I, Grossenbacher R, Noorani M,Furst P, Oncogene, 2000, 19, 2913-20. Kudoh K, Ramanna M, Ravatn R,Elkahloun A G, Bittner M L, Meltzer P S, Trent J M, Dalton W S, Chin KV, Cancer Res, 2000, 4161-6). These reports show that the analysis ofvariations in gene expression is extremely usefFul in comparison ofcharacteristics of two or more cell populations and comprehensivestudies of biological changes of cells caused by drug treatment or thelike at a molecular level.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide surrogate markers forantitumor effect of E7070 and its related compounds in cases that thecompounds are caused to act on tumor cells.

The inventors of the present invention analyzed changes in geneexpression caused when E7070 and its related compounds were caused toact on tumor cells susceptible to these anticancer agents by the DNAmicroarray method and found changes in gene expression caused by theseanticancer agents in common.

Further, they also found that, among these genes, there were genes thatexhibited changes of expression common to three kinds of cancers andthat expression changes of these genes can be used as surrogate markersfor the antitumor effect of E7070 and its related compounds, and thusaccomplished the present invention.

The present invention provides the followings.

1. A method for testing susceptibility of a tumor cell to an anticanceragent, which comprises:

1) measuring an expression level or levels of a gene or genes selectedfrom the group consisting of the genes listed in Tables 3 and 4 in atumor cell collected from a cancer patient treated with an anticanceragent represented by the following general formula (I):

wherein A represents a monocyclic or bicyclic aromatic ring, which maybe substituted,

-   B represents a 6-membered unsaturated hydrocarbon ring or a    6-membered unsaturated heterocyclic ring containing one nitrogen    atom as a heteroatom, each of which may be substituted,-   C represents a 5-membered heterocyclic ring containing one or two    nitrogen atoms, which may be substituted,-   W represents a single bond or —CH═CH—,-   X represents —N(R¹)— or an oxygen atom,-   Y represents a carbon or nitrogen atom,-   Z represents —N(R²)— or a nitrogen atom, and-   R¹ and R² may be identical or different and each represent a    hydrogen atom or a lower alkyl group, and

2) determining that the tumor cell is susceptible to the anticanceragent when the expression level or levels of the gene or genes listed inTable 3 increase, or the expression level or levels of the gene or geneslisted in Table 4 decrease in comparison with those in a tumor cellcollected from the cancer patient before the treatment with theanticancer agent.

2. A method for testing susceptibility of a tumor cell to an anticanceragent, which comprises:

1) causing an anticancer agent to act on a tumor cell collected from acancer patient, the said anticancer agent being represented by thefollowing general formula (I):

wherein A represents a monocyclic or bicyclic aromatic ring, which maybe substituted,

-   B represents a 6-membered unsaturated hydrocarbon ring or a    6-membered unsaturated heterocyclic ring containing one nitrogen    atom as a heteroatom, each of which may be substituted,-   C represents a 5-membered heterocyclic ring containing one or two    nitrogen atoms, which may be substituted,-   W represents a single bond or —CH═CH—,-   X represents —N(R¹)— or an oxygen atom,-   Y represents a carbon or nitrogen atom,-   Z represents —N(R²)— or a nitrogen atom, and-   R¹ and R² may be identical or different and each represent a    hydrogen atom or a lower alkyl group,

2) measuring an expression level or levels of a gene or genes selectedfrom the group consisting of the genes listed in Tables 3 and 4 in thetumor cell, and

3) determining that the tumor cell is susceptible to the anticanceragent when the expression level or levels of the gene or genes listed inTable 3 increase, or the expression level or levels of the gene or geneslisted in Table 4 decrease in comparison with those in an untreatedtumor cell.

3. The method according to 1 or 2, wherein the expression level orlevels of the gene or genes are measured by quantifying an RNA or RNAswhich are a transcription product or products of the gene or genes byusing a DNA microarray.

4. The method according to 1 or 2, wherein the expression level orlevels of the gene or genes are measured by quantifying an RNA or RNAswhich are a transcription product or products of the gene or genes byquantitative PCR.

5. A reagent for quantifying an RNA for use in the method as defined in4, which comprises an oligonucleotide complementary to the RNA as acomponent.

6. The method according to 1 or 2, wherein the expression level orlevels of the gene or genes are measured by quantifying a protein orproteins which are a gene product products of the gene or genes by animmunochemical method.

7. The method according to 6, wherein the expression level or levels ofthe gene or genes are measured by quantifying a protein or proteinswhich are a gene product or products of the gene or genes by ELISA.

8. The method according to 6, wherein the expression level or levels ofthe gene or genes are measured by quantifying a protein or proteinswhich are a gene product or products of the gene or genes by Westernblotting.

9. An immunoassay reagent used in the method as defined in 6, whichcomprises an antibody directed to the protein as a component.

10. The method according to 1 or 2, wherein A represents benzene orpyridine which may be substituted, B represents benzene which may besubstituted, C represents pyrrole which may be substituted, W representsa single bond, and X and Z both represent —NH—.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows cell growth inhibition curves obtained with E7070, of cellstrains HCT116-C9, HCT116-C9-C1, LX-1 and LX-1-E2.

FIG. 2 shows results of analysis of variations in gene expression in theE7070-susceptible strain HCT116-C9 by quantitative PCR.

FIG. 3 shows results of analysis of variations in gene expression in theE7070-susceptible strain LX-1 by quantitative PCR.

FIG. 4 shows results of analysis of variations in gene expression in theE7070-resistant strain HCT116-C9-C1 by quantitative PCR.

FIG. 5 shows results of analysis of variations in gene expression in theE7070-resistant strain LX-1-E2 by quantitative PCR.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereafter, embodiments of the present invention will be described indetail.

The test method of the present invention is characterized in that achange in expression level of a gene in a tumor cell caused when thetumor cell is exposed to an anticancer agent in vivo or in vitro is usedas an index of susceptibility of the tumor cell to the anticancer agent.

Therefore, the test method of the first embodiment of the presentinvention comprises the steps of 1) measuring an expression level orlevels of a gene or genes selected from the group consisting of thegenes listed in Tables 3 and 4 of a tumor cell collected from a cancerpatient treated with an anticancer agent represented by the generalformula (I), and 2) determining that the tumor cell is susceptible tothe anticancer agent when the expression level or levels of the gene orgenes listed in Table 3 increase, or the expression level or levels ofthe gene or genes listed in Table 4 decrease in comparison with those ina tumor cell collected from the cancer patient before the treatment withthe anticancer agent.

Further, the test method of the second embodiment of the presentinvention comprises the steps of 1) causing an anticancer agentrepresented by the general formula (I) to act on a tumor cell collectedfrom a cancer patient, 2) measuring an expression level or levels of agene or genes selected from the group consisting of the genes listed inTables 3 and 4 in the tumor cell, and 3) determining that the tumor cellis susceptible to the anticancer agent when the expression level orlevels of the gene or genes listed in Table 3 increase, or theexpression level or levels of the gene or genes listed in Table 4decrease in comparison with those in an untreated tumor cell.

The anticancer agent used in the present invention is a sulfonamidederivative or a sulfonnic acid ester derivative represented by thegeneral formula (I).

In the general formula (I), the “monocyclic or bicyclic aromatic ring,which may be substituted” represented by A is an aromatic hydrocarbonring or an aromatic heterocyclic ring containing at least one ofnitrogen, oxygen and sulfur atoms, each of which may have one to threesubstituents thereon. Examples of such aromatic rings included in thering A include pyrrole, pyrazole, imidazole, thiophene, furan, thiazole,oxazole, benzene, pyridine, pyrimidine, pyrazine, pyridazine,naphthalene, quinoline, isoquinoline, phthalazine, naphthyridine,quinoxaline, quinazoline, cinnoline, indole, isoindole, indolizine,indazole, benzofuran, benzothiophene, benzoxazole, benzimidazole,benzopyrazole, benzothiazole and so forth. They may have one to threesubstituents. When two or more substituents are present, they may be thesame or different. Examples of the substituents include an amino groupwhich may be substituted with a lower alkyl or lower cycloalkyl group, alower alkyl group, a lower alkoxy group, hydroxyl, nitro, mercapto,cyano, a lower alkylthio group, halogen, a group represented by theformula -a-b [wherein a represents a single bond, —(CH₂)_(k)—,—O—(CH₂)_(k)—, —S—(CH₂)_(k)— or —N(R³)—(CH₂)_(k)— (k is an integer of 1to 5, R³ represents a hydrogen atom or a lower alkyl group), and brepresents —CH₂-d (wherein d represents an amino group which may besubstituted with a lower alkyl group, halogen, hydroxyl, a loweralkylthio group, cyano or a lower alkoxy group)], a group represented bythe formula -a-e-f [wherein a has the same meaning as defined above, erepresents —S(O)— or —S(O)₂—, f represents an amino group which may besubstituted with a lower alkyl or lower alkoxy group, a lower alkylgroup, trifluoromethyl, —(CH₂)_(m)-b or —N(R⁴)—(CH₂)_(m)-b (wherein bhas the same meaning as defined above, R⁴ represents a hydrogen atom ora lower alkyl group, and m is an integer of 1 to 5)], a grouprepresented by the formula -a-g-h [wherein a has the same meaning asdefined above, g represents —C(O)— or —C(S)—, h represents an aminogroup which may be substituted with a lower alkyl group, hydroxyl, alower alkyl group, a lower alkoxy group, —(CH₂)_(n)-b or—N(R⁵)—(CH₂)_(n)-b (wherein b has the same meaning as defined above, R⁵represents a hydrogen atom or a lower alkyl group, and n is an integerof 1 to 5)], a group represented by the formula -a-N(R⁶)-g-i [wherein aand g have the same meanings as defined above, R⁶ represents a hydrogenatom or a lower alkyl group, i represents a hydrogen atom or a loweralkoxy group or f (f has the same meaning as defined above)], a grouprepresented by the formula -a-N(R⁷)-e-f (wherein a, e and f have thesame meanings as defined above, and R⁷ represents a hydrogen atom or alower alkyl group) and group represented by the formula—(CH₂)_(p)-j-(CH₂)_(q)-b (wherein j represents an oxygen or sulfur atom,b has the same meaning as defined above, and p and q may be the same ordifferent and each represent an integer of 1 to 5) and so forth.

When the substituent is an amino group substituted with two of alkylgroups, both of the alkyl groups may bond to form a 5- or 6-memberedring. Further, when A is a nitrogen-containing heterocyclic ring havinghydroxyl or mercapto, these groups may present in the form of an oxo orthioxo group by resonance.

The “6-membered unsaturated hydrocarbon ring or 6-membered unsaturatedheterocyclic ring containing one nitrogen atom as a heteroatom, whichmay be substituted” represented by B means benzene or pyridine which maybe partially hydrogenated. It may have one or two of substituents on thering, and when two of substituents are present, they may be the same ordifferent.

The “5-membered heterocyclic ring containing one or two nitrogen atoms,which may be substituted” represented by C means pyrrole, pyrazole orimidazole which may be partially hydrogenated. It may have one or two ofsubstituents on the ring, and when two of substituents are present, theymay be the same or different.

Examples of the substituents that the rings of B and C may have includehalogen, cyano, a lower alkyl group, a lower alkoxy group, hydroxyl,oxo, a group represented by the formula —C(O)-r (wherein r represents ahydrogen atom, an amino group which may be substituted with a loweralkyl group, a lower alkyl group, a lower alkoxy group or hydroxyl), anamino group substituted with a lower alkyl group, trifluoromethyl and soforth.

In the general formula (I), the lower alkyl group in the definitions ofR¹ and R² as well as the substituents that the rings of A, B and C mayhave means a linear or branched alkyl group having 1 to 6 carbon atoms,and examples thereof include methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl (amyl), isopentyl,neopentyl, tert-pentyl, 1-methylbutyl, 2-methylbutyl,1,2-dimethylpropyl, n-hexyl, isohexyl, 1-methylpentyl, 2-methylpentyl,3-methylpentyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl,1,3-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl,2-ethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl,1-ethyl-1-methylpropyl, 1-ethyl-2-methylpropyl and so forth. Amongthese, methyl, ethyl, n-propyl, isopropyl, n-butyl and isobutyl arepreferred, and methyl, ethyl, n-propyl and isopropyl are most preferred.

The lower cycloalkyl group mentioned in the definitions of thesubstituents that the ring of A may have means a cycloalkyl group having3 to 8 carbon atoms, and examples thereof include cyclopropyl,cyclopentyl, cyclohexyl and so forth.

The lower alkoxy mentioned in the definitions of the substituents thatthe rings of A, B and C may have means an alkoxyl group derived from theaforementioned lower alkyl group, such as methoxy, ethoxy, n-propoxy,isopropoxy, n-butoxy, isobutoxy and tert-butoxy. Among these, methoxyand ethoxy are most preferred. The lower alkylthio group means analkylthio group derived from the aforementioned lower alkyl group.Further, examples of the halogen include fluorine, chlorine, bromine andso forth.

The sulfonamide derivatives or sulfonic acid ester derivativesrepresented by the general formula (I) may form a salt with an acid or abase. The anticancer agent used in the present invention also includessalts of the compounds represented by the general formula (I). Examplesof the salt with an acid include salts with inorganic acids, such ashydrochlorides, hydrobromides and sulfates, and salts with organic acidssuch as acetic acid, lactic acid, succinic acid, fumaric acid, maleicacid, citric acid, benzoic acid, methanesulfonic acid andp-toluenesulfonic acid, and examples of the salt with a base includesalts with inorganic bases, such as sodium salts, potassium salts andcalcium salts and salts with organic bases such as triethylamine,arginine and lysine.

It is needless to say that the compounds include hydrates and opticalisomers of these compounds if they are present. The anticancer agentused in the present invention exhibits a high antitumor activity, andthe anticancer agents also includes compounds which undergo metabolismsuch as oxidation, reduction, hydrolysis and conjugation in vivo toexhibit the antitumor activity. Further, the anticancer agent used inthe present invention includes compounds which undergo metabolism invivo such as oxidation, reduction and hydrolysis to form compoundsrepresented by the general formula (I).

Tumor cells of which susceptibility is tested are not particularlylimited so long as they are susceptible to the anticancer agentrepresented by the general formula (I). Examples thereof include tumorcells derived from colon cancer, lung cancer, breast cancer, leukemia,pancreas cancer, kidney cancer, melanoma, malignant lymphoma, head andneck cancer, gastric cancer and so forth.

The tumor cells collected from a cancer patient include tumor cellscontained in a cancer tissue isolated from the cancer patient.

In the test method of the first embodiment, the tumor cell collectedfrom a cancer patient may be a tumor cell collected from a cancerpatient treated with an anticancer agent represented by the generalformula (I) in such an amount that susceptibility of the tumor cell canbe measured. Usually, tumor cells collected from a cancer patienttreated with a dose of 100 to 1500 mg for 1 to 14 days are used.

In the test method of the second embodiment, conditions for causing theanticancer agent represented by the general formula (I) to act on atumor cell collected from a cancer patient are not limited so long assusceptibility of the tumor cells can be measured, and culture isusually performed in a medium containing the anticancer agent at aconcentration of 0.01 to 10 μM for 6 to 72 hours.

The gene expression level can be measured by quantifying RNA as a genetranscription product or a protein as a gene product. The RNA or proteincan be quantified usually by extracting the RNA or protein from tumorcells and quantifying the RNA or protein in the extract. Hereafter,examples of 1. extraction of RNA or protein, 2. quantification of RNA,and 3. quantification of protein will be explained in detail in thisorder.

1. Extraction of RNA or Protien

-   1) Extraction of RNA or protein from cancer tissue of patient    treated with anticancer agent represented by general formula (I)

RNA or protein is extracted from a cancer tissue collected by biopsy orthe like from a patient treated with an anticancer agent represented bythe general formula (I) by the method described below.

RNA can be extracted according to a usual RNA extraction method. Forexample, extraction can be carried out by using TRIZOL Reagent (LifeTechnologies Oriental) or the like according to the attached operationmanual. Specifically, a cancer tissue with 1 ml of TRIZOL reagent per 50to 100 mg of the cancer tissue is homogenized by using a Teflonhomogenizer. The mixture is centrifuged (12,000×g, 10 minutes, 4° C.),and the obtained supernatant is left at room temperature for 5 minutesand 0.2 ml of chloroform is added thereto per 1 ml of the used TRIZOLReagent. The resulting solution is vigorously stirred by shaking for 15seconds, left at room temperature for 2 to 3 minutes and thencentrifuged (12,000×g, 15 minutes, 4° C.). After the centrifugation, theaqueous layer is transferred to a new tube, and 0.5 ml of isopropylalcohol is added per 1 ml of the used TRIZOL reagent. The mixture isleft at room temperature for 10 minutes and centrifuged (12,000×g, 10minutes, 4° C.). The obtained precipitates are washed with 75% ethanol,air-died and used in subsequent operations as total RNA.

Protein can be extracted from a cancer tissue according to the methodsdescribed in Bollag, D. M., Rozycki M. D., Edelstein S. J., ProteinMethods, 1996, Wiley-Liss, Inc., New York, U.S.A.; Walker, J. M., Theprotein handbook, 1996, Humana Press, New Jersey, U.S.A. and so forth.

-   2) Extraction of RNA or protein from cancer cell cultured in the    presence of anticancer agent represented by general formula (I)

Cancer cells (tumor cells) are isolated in a conventional manner from acancer tissue obtained from a patient by biopsy or the like. Forexample, according to the method of Hamburger et al. (Hamburger A.,Salmon S. E., Kim M. B., Trent J. M., Soehnlen B. J., Alberts D. S. andSchmidt H. T., Cancer Res., 38, 3438-3443, 1978), the obtained tissue isaseptically chopped, and then a cell suspension is prepared by using astainless mesh, injection needle, nylon mesh or the like. The obtainedcells are cultured in a suitable medium (for example, RPMI-1640, MEM,McCoy medium etc. containing 10-15% FCS). The obtained cancer cells arecultured at 37° C. in the presence of the anticancer agent representedby the general formula (I) under 5% CO₂ condition for a suitable period,preferably, for 3, 6, 12 or 24 hours, more preferably for 12 hours, andRNA or protein is extracted by the method described below. Further, itis also possible to specifically isolate only cancer cells from a tissueobtained by biopsy or the like by using the soft agar culture method(Hamburger A., and Salmon S. E., Science, 197, 461-463, 1977; HamburgerA., and Salmon S. E., J. Clin. Invest., 60, 846-854, 1977; Von Hoff D.D., and Johnson, G. E., Proc. Am. Assoc. Cancer Res., 20, 51, 1979) anduse them.

RNA can be extracted from a cancer cell according to a usual RNAextraction method as the extraction of RNA from a cancer tissue. Forexample, when TRIZOL Reagent (Life Technologies Oriental) is used,extraction can be carried out according to the attached operationmanual. Specifically, 1 ml of TRIZOL Reagent is added per 5 to 10×10⁶cancer cells, and the same operations as in the extraction of RNA from acancer tissue can be performed.

Extraction of protein from cancer cells can also be carried outaccording to methods described in publications as in the extraction fromcancer tissue.

2. Quantification of RNA

RNA can be quantified by techniques of Northern blotting analysis, DNAmicroarray analysis, RT-PCR, quantitative PCR and so forth. It ispreferable to use a DNA microarray or quantitative PCR. These techniqueswill be explained below, but the present invention is not limited tothem.

Quantification by using a DNA microarray is performed as follows. First,double-stranded cDNA is synthesized by using the obtained RNA as atemplate, SuperScript Choice System (Life Technologies Oriental) andT7-d(T)₂₄ primer. Subsequently, biotinylated cRNA is synthesized byusing the cDNA as a template.

Specifically, single-stranded DNA is first synthesized from the obtainedRNA by using the T7-d(T)₂₄ primer. Then, dNTP, DNA ligase, DNApolymerase I and RNase H are added thereto to react them and T4 DNApolymerase I is further added thereto to complete the synthesis ofdouble-stranded cDNA. The obtained cDNA is purified, and thenbiotinylated UTP and CTP are added to allow a labeling reaction by usingRNA Transcript Labeling Kit (Enzo Diagnostics). The reaction product ispurified and heated at 94° C. in 200 mM Tris/acetate (pH 8.1), 150 mMmagnesium acetate, 50 mM potassium acetate for 35 minutes to obtainfragmented cRNA.

The fragmented cRNA is hybridized with GeneChip Hu6800 (Affymetrix) oran update product, for example, in 100 mM MES, 1 M sodium salt, 20 mMEDTA, 0.01% Tween 20 at 45° C. for 16 hours. After the hybridization,the GeneChip is washed and stained according to the protocol EukGE-WS2attached to the Affymetrix Fluidics Station. For the staining,streptavidin-phycoerythrin and biotinylated goat anti-streptavidinantibody are used. The stained GeneChip is scanned by using HP Argon IonLaser Confocal Microscope (Hewlett Packard) to measure its fluorescenceintensity. When this fluorochrome is used, fluorescence is measured at570 nm by using an excitation light at 488 nm.

The quantitative data analysis is preferably conducted by using GeneChipSoftware (Affymetrix). To quantify RNA, an average of “differences[(Perfect match hybridization signal)−(Mismatch signal)]” is obtainedfor each probe family. When this value is 50 or higher, and quantitativevalues of RNA obtained under two kinds of conditions diverge, preferablywhen they diverge 1.8 times or more, it is determined that the geneexpression has significantly “increased” or “decreased”.

When the RNA level or levels of the gene or genes listed in Table 3increase, or the RNA level or levels of the gene or genes listed inTable 4 decrease, it is determined that the tumor cells are susceptibleto the anticancer agent.

Further, quantitative PCR is performed as follows by using SYBR Greenand ABI Prism 7700 Sequence Detection System (Perkin-Elmer AppliedBiosystems).

The operation is performed in two steps of a reverse transcriptionreaction and PCR reaction. The reverse transcription reaction as thefirst step is carried out by adding dNTP, oligo d(T)₁₆ primer, RNaseinhibitor mixture and Multiscribe Reverse Transcriptase (Perkin-ElmerApplied Biosystems) to the obtained RNA, keeping the mixture at 25° C.for 10 minutes and then heating the mixture at 48° C. for 30 minutes.The reaction is terminated by heating at 95° C. for 5 minutes.

The obtained cDNA is subjected to the PCR reaction as the second step.The PCR reaction is carried out, for example, in a reaction systemcomposed of 4 ng of cDNA, 1×SYBR PCR buffer, 3 mM MgCl₂, 200 μM each ofdATP, dCTP and dGTP, 400 μM dUTP, 200 nM primer pair, 0.01 U/μl AmpEraseUNG and 0.025 U/μl AmpliTaq Gold DNA Polymerase (Perkin-Elmer AppliedBiosystems). The PCR reaction is performed with, for example, reactionsat 50° C. for 2 minutes and at 95° C. for 10 minutes followed by a cycleof reactions at 95° C. for 20 seconds, at 55° C. for 20 seconds and at72° C. for 30 seconds, which are repeated 40 times. The primers and theprobe are designed, for example, by using Primer Expression(Perkin-Elmer Applied Biosystems). For comparison of two or morespecimens, a quantified value is corrected into transcription amount ineach specimen by using mRNA level of a house keeping gene, which showslittle variation in transcription amount, preferably mRNA level ofGAPDH, and used.

When the RNA level or levels of the gene or genes listed in Table 3increase, or the RNA level or levels of the gene or genes listed inTable 4 decrease, it is determined that the tumor cells are susceptibleto the anticancer agent.

The present invention also provides a reagent for quantifying RNA foruse in the test method of the present invention, which comprises anoligonucleotide complementary to RNA as a transcription product of agene of which expression level is to be measured, as a component. Theoligonucleotide included as a component is a primer and/or probe used inquantitative PCR and can be designed as described above. TheRNA-quantifying reagent of the present invention may contain componentsconventionally used in an ordinary quantifying reagent in addition tothe above oligonucleotide.

3. Quantification of Protein

Although protein is quantified based on its activity or antigenicity,quantification based on antigenicity, i.e., immunochemicalquantification, which can be easily and generally applied to proteins,is preferred.

As for antibody directed to a protein, an antigen determinant ispredicted from the amino acid sequence based on the report of Parker etal. (Parker J. M. R., Guo D., Hodges R. S., Biochemistry, 25, 5425,1986) or the report of Karplus et al. (Karplus P. A., Schulz G. E.,Naturwissenschaften, 72, 212, 1985) to synthesize a peptide.Alternatively, a fusion protein, for example, a fusion protein withglutathione synthetase (GST) is expressed and purified by using aglutathione column to produce an antigen, and a rabbit, a mouse or thelike is immunized with the obtained antigen to produce polyclonal orpreferably monoclonal antibodies (Harlow E., Lane D., Antibodies: ALaboratory Manual, 1988, Cold Spring Harbor Laboratory Press, New York).Further, when commercially available antibodies are available, they canalso be used after their specificity is confirmed.

By using the obtained antibodies, ELISA or RIA is performed to quantifythe protein, for example, by the enzyme immunoassay method (Ishikawa E.et al., Igakushoin, 1982) or the method described in Ishikawa E., KawaiT., Miyai, K., Enzyme Immunoassay, Igakushoin, Tokyo, New York, 1981.When the protein is secreted out of the tumor cell, the protein in amedium can be quantified without extracting the protein from the tumorcell.

When the protein level or levels of the gene or genes listed in Table 3increase, or the protein level or levels of the gene or genes listed inTable 4 decrease, it is determined that the tumor cells are susceptibleto the anticancer agent.

The present invention also provides an immunoassay reagent for use inthe test method of the present invention, which comprises an antibodydirected to the protein as a component. The antibody used as a componentcan be obtained as described above. The immunoassay reagent of thepresent invention may contain components conventionally used in ordinaryimmunoassay reagents in addition to the aforementioned antibody.

EXAMPLES

Hereafter, the present invention will be described more specificallywith reference to the following specific examples. However, the presentinvention is not limited to them.

Example 1 Culture of E7070-Susceptible and E7070-Resistant Strains andExtraction of RNA

All cells were cultured in RPMI-1640 medium supplemented with 10% offetal bovine serum, 100 units/ml of penicillin and 100 μg/ml ofstreptomycin at 37° C. under 5% CO₂.

E7070, or ER-35748 or ER-68487 represented by the structural formulashown below was added to the media of an E7070-susceptible strainHCT116-C9 and an E7070-resistant strain HCT116-C9-C1 at a concentrationof 8 μM and the cells were cultured. Then, cells were recovered after 0,3, 6 and 12 hours. Further, the cells were also cultured for 12 hourswithout adding the drug and recovered.

Total RNA was extracted from these cells and subjected to the subsequentanalyses. The RNA extracted from the cells cultured for 12 hours withthe drug and the RNA from extracted the cells cultured for 12 hourswithout the drug were used for the gene expression analysis using a DNAmicroarray described in Example 2. HCT116-C9 was a substrain isolatedfrom HCT116 derived from human colon cancer (American Type CultureCollection, Manassas, Va., U.S.A.), and HCT116-C9-C1 was anE7070-resistant substrain obtained by culturing this HCT116-C9 in thepresence of E7070 with gradually increasing the E7070 concentration.

Similarly, E7070 was added to the media of an E7070-susceptible strainLX-1 and an E7070-resistant strain LX-1-E2 at a concentration of 8 μMand the cells were cultured. Cells were recovered after 0, 3, 6 and 12hours. Total RNA was extracted from these cells and subjected to thesubsequent analyses. LX-1 (Cancer Chemotherapy Center, Japan Foundationfor Cancer Research, Tokyo, Japan) was a cell strain derived from humanparvicellular lung cancer, and LX-1-E2 is an E7070-resistant substrainobtained by culturing this LX-1 in the presence of E7070 with graduallyincreasing the E7070 concentration.

The used cell strains HCT116-C9, HCT116-C9-C1, LX-1 and LX-1-E2 werecultured for 72 hours with addition of E7070. Cell growth inhibitioncurves measured by the MTT method (Mosmann T., J. Immunol. Methods, 65,55 (1983)) are shown in FIG. 1. The actual operation was performed byusing CellTiter 96 Non-Radioactive Cell Proliferation Assay (Promega,Madison, Wis.) according to the attached operation manual.

Total RNA was extracted from the recovered cells by using TRIZOL Reagent(Life Technologies Oriental) according to the attached operation manual.

Example 2 Gene Expression Analysis Using DNA Microarray

1) Synthesis of cDNA and Biotinylated cRNA

Each RNA obtained in Example 1 was dissolved in 100 μl of sterilizedwater treated with diethyl pyrocarbonate (DEPC) and further purified byusing RNeasy Column (QIAGEN), and double-stranded cDNA was synthesizedby using SuperScript Choice System (Life Technologies Oriental) and aT7-d(T)₂₄ primer.

First, to 10 μg of RNA, 5 μM T7-d(T)₂₄ primer, 1×First strand buffer, 10mM DTT, 500 μM dNTP mix and 20 units/μl of SuperScript II ReverseTranscriptase were added and the mixture was allowed to react at 42° C.for 1 hour to synthesize single-stranded DNA. Subsequently, to the DNA,1×Second strand buffer, 200 μM dNTP mix, 67 U/ml of DNA ligase, 270 U/mlof DNA polymerase I and 13 U/ml of RNase H were added and the mixturewas allowed to react at 16° C. for 2 hours to synthesize double-strandedcDNA. Further, to the cDNA, 67 U/ml of T4 DNA polymerase I was added andthe mixture was allowed to react at 16° C. for 5 minutes and then 10 μlof 0.5 M EDTA was added to terminate the reaction.

The obtained cDNA was purified with phenol/chloroform, and a labelingreaction with biotinylated UTP and CTP was carried out by using RNATranscript Labeling Kit (Enzo Diagnostics) according to the attachedoperation manual. The reaction product was purified with Rneasy Columnand heated in 200 mM Tris/acetate buffer (pH 8.1), 150 mM magnesiumacetate, 50 mM potassium acetate at 94° C. for 35 minutes to obtainfragment cRNA.

2) Hybridization with DNA Microarray (GeneChip) and Measurement

The fragmented cRNA was hybridized with GeneChip Hu6800 (Affymetrix) in100 mM MES, 1 M sodium salt, 20 mM EDTA and 0.01% Tween 20 at 45° C. for16 hours. After the hybridization, the GeneChip was washed and stainedaccording to the protocol EukGE-WS2 attached to Affymetrix FluidicsStation. For the staining, streptavidin-phycoerythrin and biotinylatedgoat anti-streptavidin antibody were used. The stained GeneChip wasscanned by using HP Argon Ion Laser Confocal Scanner (Hewlett Packard)to measure its fluorescence intensity. The fluorescence was measured at570 nm by using an excitation light at 488 nm.

The quantitative data analyses were all conducted by using GeneChipSoftware (Affymetrix). To quantify RNA, an average of “differences[(Perfect match hybridization signal)−(Mismatch signal)]” was obtainedfor each probe family. When this value was 50 or higher, andquantitative values of RNA measured under two kinds of conditionsdiverge, preferably they diverge 1.8 times or more, it was determinedthat the gene expression had significantly “increased” or “decreased”.

E7070, or ER-35748 or ER-68487 was added to a medium for HCT116-C9 at aconcentration of 8 μM and culture was performed. The results of analysisof RNA extracted from the cells at 12 hours by using GeneChip werecompared with the results of RNA analysis for HCT116-C9 cells obtainedin the same manner except for not adding the drug. As a result, a listof genes of which expression levels were increased in common by thetreatments with three types of drugs, E7070, ER-35748 and ER-68487(GenBank registration numbers are also shown (the same shall apply inother tables shown below)) is shown in Table 1. Similarly, a list ofgenes of which expression levels were decreased in common by thetreatments with the three types of drugs is shown in Table 2.

TABLE 1 Registration number Gene name D59253 NCBP interacting protein 1D78514 Ubiquitin-conjugating enzyme D83767 Rep-8 HG1139-HT4910FK506-Binding protein HG3484-HT3678 Cdc-like kinase 1 (Clk1) J04152Tumor-associated antigen GA733-1, M1S1 M21154 S-adenosylmethioninedecarboxylase M60724 p70 Ribosomal S6 kinase alpha-1 M84349Transmembrane protein CD59 S61953 c-ErbB3 receptor tyrosine kinaseU52960 RNA polymerase II complex component SRB7 U84720 Export proteinRAE1 U92014 Defective mariner transposon Hsmar2 Z18951 Caveolin

TABLE 2 Registration number Gene name AB000409 Serine/threonine proteinkinase MNK1 AB000450 Putative serine/threonine protein kinase VRK2AB002380 Leukemia-associated Rho GEF AB003102 Proteasome 26S subunitp44.5 AC002045 CIT987SK-A-589H1 AF002020 Niemann-Pick C disease protein(NPC1) AF008445 Phospholipid scramblase D00723 Hydrogen carrier protein,glycine synthase D14659 KIAA0103 D21852 KIAA0029 D26535 Dihydrolipoamidesuccinyltransferase D28364 Annexin II D29677 KIAA0054 D29810 Unknownproduct D29956 Ubiquitin specific protease 8 D30756 KIAA0049 D31883Actin-binding LIM protein D32002 Nuclear cap binding protein D38521KIAA0077 D38552 KIAA0073 D38553 KIAA0074 D43947 KIAA0100 D43948 ch-TOGD50645 SDF2 D50663 Dynein (TCTEL1) D50912 RNA-binding motif protein 10D50916 Ubiquitination factor E4A D61391 PAP39 D63480 KIAA0146 D63506Syntaxin-binding protein 3 D63875 TPR-containing/SH2-binding proteinD63880 KIAA0159 D78586 Multifunctional protein CAD D79983 KIAA0161D79987 KIAA0165 D79988 KIAA0166 D79991 KIAA0169 D83776 KIAA0191 D83781KIAA0197 D84307 Phosphoethanolamine cytidylyltransferase D86981 Amyloidprecursor protein-binding protein 2 D87435 KIAA0248 D87446 KIAA0257D87448 DNA topoisomerase II-binding protein D87743 Solute carrier family9 HG1869-HT1904 Male enhanced antigen HG2379-HT3996 Serinehydroxymethyltransferase, cytosolic HG4094-HT4364 Transcription factorLsf-Id J04088 DNA topoisomerase II (top2) J04501 Muscle glycogensynthase J04543 Synexin L06845 Cysteinyl-tRNA synthetase L07033Hydroxymethylglutaryl-CoA lyase L07540 Replication factor C, 36-kDasubunit L07597 Ribosomal protein S6 kinase 2 L07758 IEF SSP 9502 L21936Succinate dehydrogenase flavoprotein subunit L25444 TAFII70-alpha L25931Lamin B receptor L33075 Ras GTPase-activating-like protein IQGAP1 L33881Protein kinase C iota isoform L38810 Proteasome 26S subunit p45 L40395Eukaryotic initiation factor 2B-beta L41870 Retinoblastomasusceptibility protein (RB1) L47276 Alpha topoisomerase truncated-formM15796 PCNA M19267 Tropomyosin M22632 Mitochondrial aspartateaminotransferase M23379 GTPase-activating protein ras p21 M24486 Prolyl4-hydroxylase alpha subunit M29204 DNA-binding factor M29550 CalcineurinA1 M30496 Ubiquitin carboxyl-terminal hydrolase M33518 HLA-B-associatedtranscript 2 (BAT2) M34309 Epidermal growth factor receptor HER3 M37400Cytosolic aspartate aminotransferase M55905 Mitochondrial NAD(P) +dependent malic enzyme M58525 Catechol-O-methyltransferase M59911Integrin alpha-3 M61764 Gamma-tubulin M62994 Filamin B M74089 TB1 M85085Cleavage stimulation factor M86707 Myristoyl CoA: protein N-myristoyltransferase M87338 Replication factor C, 40-kDa subunit (A1) M87339Replication factor C, 37-kDa subunit (RFC4) M88163 Global transcriptionactivator (hSNF2/SWI2) M91432 Medium-chain acyl-CoA dehydrogenase (MCAD)M92439 Leucine-rich protein M93056 Monocyte/neutrophil elastaseinhibitor M95809 Basic transcription factor 62 kD subunit (BTF2) M95929Homeobox protein PHOX1 M97935 Transcription factor ISGF-3 S58544 75 kDainfertility-related sperm protein S59184 RYK = related to receptortyrosine kinase S72904 APK1 antigen = MAb KI recognized S78085 PDCD2,Rp8 homolog S80343 Arginyl-tRNA synthetase (ArgRS) U01062 Inositol1,4,5-triphosphate receptor type 3 U02566 Receptor tyrosine kinase tifU04285 Lysosomal acid lipase U06631 H326 U07231 G-rich sequence factor-1U07681 Isocitrate dehydrogenase 3 (NAD+) alpha U07919 Aldehydedehydrogenase 6 (ALDH6) U08815 Splicesomal protein SAP61 U10324 Nuclearfactor NF90 U11791 Cyclin H U15174 Nip3 U15306 DNA-binding protein NFX1U18291 CDC16Hs U18934 Receptor tyrosine kinase DTK U20979 Chromatinassembly factor-I p150 subunit U22233 Methylthioadenosine phosphorylaseU23028 Eukaryotic initiation factor 2B-epsilon U23946 LUCA15 U26648Syntaxin 5A U27459 Origin recognition complex protein 2 homolog U27460Uridine diphosphoglucose pyrophosphorylase U28413 Cockayne syndromecomplementation group A U28811 Cystein-rich fibroblast growth factorreceptor U28831 Immuno-reactive with anti-PTH Ab U28963 Gps2 U30313Diadenosine tetraphosphatase U30521 P311 HUM −3.1 U30827 Splicing factorSRp40-3 (SRp40) U30828 Splicing factor SRp55-2 (SRp55) U34252Gamma-aminobutyraldehyde dehydrogenase U34683 Glutathione synthetaseU36341 SLC6A8 U40282 Integrin-linked kinase U46006 Smooth muscle LIMprotein (h-SmLIM) U49844 FRAP-related protein (FRP1/ATR) U50078 Guaninenucleotide exchange factor p532 U50939 Amyloid precursor protein-bindingprotein 1 U53468 NADH: ubiquinone oxidoreductase subunit B13 U57629Retinitis pigmentosa GTPase regulator U60808 CDP-diacylglycerol synthaseU61145 Enhancer of zeste homolog 2 U61263 Acetolactate synthase homologU63743 Mitotic centromere-associated kinesin (MCAK) U65785Oxygen-regulated protein ORP150 U72514 C2f U72515 C3f U76764 CD97 U77413O-linked GlcNAc transferase U77949 Cdc6-related protein (HsCDC6) U79241Clone 23759 U80034 Mitochondrial intermediate peptidase precursor U81554CaM kinase II isoform U85611 DNA-PK interaction protein (KIP) U89606Pyridoxal kinase U90426 Nuclear RNA helicase U94319 Transcriptioncoactivator p75 (DFS70) U95740 362G6.1 U97188 Putative RNA bindingprotein KOC X06745 DNA polymerase alpha subunit X13482 U2 snRNP-specificA protein X51956 Neuron specific (gamma) enolase X53587 Integrin beta-4X54199 GARS-AIRS-GART X54867 NKG2-A X59871 T cell factor 1 X61100 75 kDasubunit NADH dehydrogenase precursor X66364 Serine/threonine proteinkinase PSSALRE X68836 S-adenosylmethionine synthetase X70476 Subunit ofcoatomer complex X75535 PxF X81003 HCG V X84740 DNA ligase III X94754Yeast methionyl-tRNA synthetase homologue X98248 Sortilin X99209Arginine methyltransferase Y08612 Nup88 Y08682 Carnitinepalmitoyltransferase I type I Y13115 Serine/threonine protein kinase SAKZ11518 Histidyl-tRNA synthetase Z17227 Transmenbrane receptor proteinCRF2-4 Z46629 SOX9 Z68747 Imogen 38

Two kinds of E7070-resistant strains (C9-C1 and another resistant strain(C9-C4) obtained in the same method as C9-C1) were treated with 8 μM ofE7070 for 12 hours in the same manner as described above, and variationsof the gene expression were analyzed by using GeneChip for the geneslisted in Tables 1 and 2. As a result, no gene that increased ordecreased 1.8 times or more was observed in the both strains.

From the above results, it is apparent that genes of which expressionlevels are changed in common by the treatments with these three types ofanticancer agents can be used as surrogate markers of antitumor effectof E7070 and its related compounds by measuring each of their changes orchanges of two or more of them in combination.

Example 3 Gene Expression Analysis by Quantitative PCR

In order to confirm that variations in gene expression obtained inExample 2 reflected the susceptibility of a tumor cell to E7070, changesof gene expression in E7070-susceptible cells and E7070-resistant cellswere examined by quantitative PCR using RNA shown in Example 1, SYBRGreen and ABI Prism 7700 Sequence Detection System (Perkin-Elmer AppliedBiosystems).

The operation was performed in two steps of a reverse transcriptionreaction and PCR reaction. The reverse transcription reaction as thefirst step was carried out by adding 1×TaqMan RT buffer, 5.5 mM MgCl₂,500 μM dNTP mix, 2.5 μM oligo d(T)₁₆ primer, 0.4 U/μl of RNase inhibitorand 1.25 U/μl of Multiscribe Reverse Transcriptase (Perkin-Elmer AppliedBiosystems) to 1 μg of the total RNA, keeping the mixture at 25° C. for10 minutes and then heating it at 48° C. for 30 minutes. The reactionwas terminated by heating at 95° C. for 5 minutes.

The obtained cDNA was subjected to the PCR reaction as the second step.The PCR reaction was carried out in a reaction system composed of 4 ngof cDNA, 1×SYBR PCR buffer, 3 mM MgCl₂, 200 μM each of dATP, dCTP anddGTP, 400 μM dUTP, 200 nM primer pair, 0.01 U/μl AmpErase UNG and 0.025U/μl AmpliTaq Gold DNA Polymerase (Perkin-Elmer Applied Biosystems). ThePCR reaction was performed with reactions at 50° C. for 2 minutes and at95° C. for 10 minutes followed by a cycle of reactions at 95° C. for 20seconds, at 55° C. for 20 seconds and at 72° C. for 30 seconds, whichare repeated 40 times. As the primers, oligonucleotides having thenucleotide sequences of SEQ ID NOS: 1 and 2 were used for GAPDH, thoseof SEQ ID NOS: 3 and 4 for S80343, those of SEQ ID NOS: 5 and 6 forU07919, those of SEQ ID NOS: 7 and 8 for U11791, those of SEQ ID NOS: 9and 10 for M95809, those of SEQ ID NOS: 11 and 12 for U18291, those ofSEQ ID NOS: 13 and 14 for U63743, and those of SEQ ID NOS: 15 and 16 forM61764.

The mRNA level in each specimen was quantified by measuring fluorescenceintensity. In comparison of two or more specimens, quantitative valueswere corrected based on the mRNA level of GAPDH in each specimen. Therelationships between the drug treatment time and each mRNA level areshown in FIGS. 2 to 5, where the quantified value at 0 hour after thedrug treatment was used as a control (100%). The genes showed thelargest expression variation in HCT116-C9 with the highestsusceptibility (FIG. 2), and in the susceptible strain LX-1, all thegenes except for S80343 showed expression variations although thevariation magnitudes were smaller than those in HCT116-C9 (FIG. 3). Onthe other hand, no expression variation was observed for the resistantstrain HCT116-C9-C1 (FIG. 4), and in the resistant strain LX-1-E2, whichshowed some growth inhibitory effect at the drug concentration of 8 μMused in the experiment, U11791 showed an expression change and M95809and U18291 showed a slight expression change (FIG. 5).

The variations in expression of the gene retrieved in Example 2correlated to the susceptibility to E7070, and it was confirmed that thegenes retrieved in Example 2 could be used solely or in combination assurrogate markers of antitumor effect of E7070 and its relatedcompounds.

Example 4 Analysis by ELISA

Among the genes listed in Table 2 in Example 2, the expression level ofX51956 (Human ENO2 gene for neuron specific (gamma) enolase), which hasbeen reported to be secreted out of the cell, was examined by thealready reported ELISA method (Duncan M. E., McAleese S. M., Booth N.A., Melvin W. T., and Fothergill J. E., J. of Immunol. Methods, 151,227-236, 1992, Yamaguchi K., Aoyagi K., Urakami K., Fukutani T., MakiN., Yamamoto S., Otsubo K., Miyake Y., and Kodama T., Jpn. J. CancerRes., 86, 698-705, 1995). The actual measurement was carried out byusing NSE ELISA Kit produced by Eiken Chemical (Tokyo) according to theattached reference.

Example 5 Gene Expression Analysis in Three Kinds Cancers

Gene expression changes in three kinds of cancers of HCT116-C9 (humancolon cancer cell strain), MDA-MB-435 (human breast cancer cell strain)and MOLT-4 (human T lymphoblastic leukemia cell strain) caused by E7070(treated with 8 μM for 12 hours) were examined by using GeneChip in thesame manner as in Example 2.

A list of the genes of which expression was enhanced 1.8 times or morein common in the three kinds of cancers is shown in Table 3. Further, alist of the genes of which expression was inhibited 1.8 times or more incommon in the three kinds of cancers is shown in Table 4. Since thegenes of which expression varies (enhanced or reduced) in common inthese strains of the three kinds of cancers established from cancerpatients with totally different genetic backgrounds are very likely tobe more closely associated with the E7070 antitumor action mechanismcommon to the tumor cells, it is considered that they can be used asvalid markers when susceptibility of a tumor cell to E7070 and itsrelated compounds is tested.

TABLE 3 Registration number Gene name D78514 Ubiquitin-conjugatingenzyme D83767 Rep-8 HG1139-HT4910 Fk506-Binding protein HG3484-HT3678Cdc-like kinase 1 (Clk1) M21154 S-adenosylmethionine decarboxylaseM60724 p70 Ribosomal S6 kinase alpha-1 S61953 c-ErbB3 receptor tyrosinekinase U52960 RNA polymerase II complex component SRB7 U84720 Exportprotein RAE1 U92014 Defective mariner transposon Hsmar2

TABLE 4 Registration number Gene name AB000409 Serine/threonine proteinkinase MNK1 AB000450 Putative serine/threonine protein kinase VRK2AC002045 CIT987SK-A-589H1 AF008445 Phospholipid scramblase D00723Hydrogen carrier protein, glycine synthase D14659 KIAA0103 D21852KIAA0029 D26535 Dihydrolipoamide succinyltransferase D32002 Nuclear capbinding protein D38521 KIAA0077 D38553 KIAA0074 D43947 KIAA0100 D50912RNA-binding motif protein 10 D61391 PAP39 D63480 KIAA0146 D63880KIAA0159 D78586 Multifunctional protein CAD D79983 KIAA0161 D79991KIAA0169 D83781 KIAA0197 D84307 Phosphoethanolamine cytidylyltransferaseD87446 KIAA0257 HG1869-HT1904 Male enhanced antigen HG2379-HT3996 Serinehydroxymethyltransferase, cytosolic HG4094-HT4364 Transcription factorLsf-Id J04088 DNA topoisomerase II (top2) L07758 IEF SSP 9502 L21936Succinate dehydrogenase flavoprotein subunit L25931 Lamin B receptorL38810 Proteasome 26S subunit p45 L41870 Retinoblastoma susceptibilityprotein (RB1) L47276 Alpha topoisomerase truncated-form M19267Tropomyosin M22632 Mitochondrial aspartate aminotransferase M29204DNA-binding factor M29550 Calcineurin A1 M30496 Ubiquitincarboxyl-terminal hydrolase M34309 Epidermal growth factor receptor HER3M55905 Mitochondrial NAD(P) + dependent malic enzyme M61764Gamma-tubulin M85085 Cleavage stimulation factor M87338 Replicationfactor C, 40-kDa subunit (A1) M91432 Medium-chain acyl-CoA dehydrogenase(MCAD) M92439 Leucine-rich protein M93056 Monocyte/neutrophil elastaseinhibitor M95809 Basic transcription factor 62 kD subunit (BTF2) M97935Transcription factor ISGF-3 S72904 APK1 antigen = MAb KI recognizedS78085 PDCD2, Rp8 homolog U01062 Inositol 1,4,5-triphosphate receptortype 3 U07231 G-rich sequence factor-1 U08815 Splicesomal protein SAP61U11791 Cyclin H U15306 DNA-binding protein NFX1 U18291 CDC16Hs U18934Receptor tyrosine kinase DTK U20979 Chromatin assembly factor-I p150subunit U22233 Methylthioadenosine phosphorylase U23028 Eukaryoticinitiation factor 2B-epsilon U23946 LUCA15 U28831 Immuno-reactive withanti-PTH Ab U28963 Gps2 U30828 Splicing factor SRp55-2 (SRp55) U34683Glutathione synthetase U40282 Integrin-linked kinase U49844 FRAP-relatedprotein (FRP1/ATR) U50939 Amyloid precursor protein-binding protein 1U57629 Retinitis pigmentosa GTPase regulator U61145 Enhancer of zestehomolog 2 U72514 C2f U77413 O-linked GlcNAc transferase U77949Cdc6-related protein (HsCDC6) U79241 Clone 23759 U80034 Mitochondrialintermediate peptidase precursor U81554 CaM kinase II isoform U89606Pyridoxal kinase U94319 Transcription coactivator p75 (DFS70) X06745 DNApolymerase alpha subunit X13482 U2 snRKP-specific A protein X51956Neuron specific (gamma) enolase X54199 GARS-AIRS-GART X61100 75 kDasubunit NADH dehydrogenase precursor X68836 S-adenosylmethioninesynthetase X70476 Subunit of coatomer complex X75535 PxF X99209 Argininemethyltransferase Y08612 Nup88 Y08682 Carnitine palmitoyltransferase Itype I Z17227 Transmembrane receptor protein CRF2-4

INDUSTRIAL APPLICABILITY

According to the present invention, by measuring an expression level orlevels of a gene or genes listed in Tables 3 and 4 in a tumor cellcollected from a cancer patient treated with an anticancer agentrepresented by the general formula (I) or causing the anticancer agentrepresented by the general formula (I) to act on a tumor cell collectedfrom a cancer patient and measuring an expression level or levels of agene or genes listed in Tables 3 and 4, susceptibility of the tumor cellto the anticancer agent can be examined.

1. A method for testing susceptibility of a tumor cell to an anticanceragent, which comprises: 1) measuring an expression level or levels of agene or genes selected from the group consisting of the genesglutathione synthetase (GenBank Accession No. U34683:gggagaaccgttcgcggaggaaaggcgaactagtgttgggatggccaccaactgggggagcctcttgcaggataaacagcagctagaggagctggcacggcaggccgtggaccgggccctggctgagggagtattgctgaggacctcacaggagcccacttcctcggaggtggtgagctatgccccattcacgctcttcccctcactggtccccagtgccctgctggagcaagcctatgctgtgcagatggacttcaacctgctagtggatgctgtcagccagaacgctgccttcctggagcaaactctttccagcaccatcaaacaggatgactttaccgctcgtctctttgacatccacaagcaagtcctaaaagagggcattgcccagactgtgttcctgggcctgaatcgctcagactacatgttccagcgcagcgcagatggctccccagccctgaaacagatcgaaatcaacaccatctctgccagctttgggggcctggcctcccggaccccagctgtgcaccgacatgttctcagtgtcctgagtaagaccaaagaagctggcaagatcctctctaataatcccagcaagggactggccctgggaattgccaaagcctgggagctctacggctcacccaatgctctggtgctactgattgctcaagagaaggaaagaaacatatttgaccagcgtgccatagagaatgagctactggccaggaacatccatgtgatccgacgaacatttgaagatatctctgaaaaggggtctctggaccaagaccgaaggctgtttgtggatggccaggaaattgctgtggtttacttccgggatggctacatgcctcgtcagtacagtctacagaattgggaagcacgtctactgctggagaggtcacatgctgccaagtgcccagacattgccacccagctggctgggactaagaaggtgcagcaggagctaagcaggccgggcatgctggagatgttgctccctggccagcctgaggctgtggcccgcctccgcgccacctttgctggcctctactcactggatgtgggtgaagaaggggaccaggccatcgccgaggcccttgctgcccctagccggtttgtgctaaagccccagagagagggtggaggtaacaacctatatggggaggaaatggtacaggccctgaaacagctgaaggacagtgaggagagggcctcctacatcctcatggagaagatcgaacctgagccttttgagaattgcctgctacggcctggcagccctgcccgagtggtccagtgcatttcagagctgggcatctttggggtctatgtcaggcaggaaaagacactcgtgatgaacaagcacgtggggcatctacttcgaaccaaagccatcgagcatgcagatggtggtgtggcagcgggagtggcagtcctggacaacccataccctgtgtgagggcacaaccaggccacgggaccttctatcctctgtatttgtcattcctctcctagccctcctgaggggtatcctcctaaagacctccaaagtttttatggaagggtaaatactggtaccttcccccagctttccatctgaggaccagaaaagttgtgtctcccttagatgagatctagacgcccccaaatccttgagatgtgggtatagctcagggtaagctgctctgaggtaaaggtccatgaaccctgccccactcctgtcagcccctcatcagccttttcagcaggttccagtgcctgacttgggataggactgagtggtaggaggagggggagtggaggggcatagcctttccctaattctgccttaaataaaactgcattgctgt (SEQ ID NO: 17)), mitochondrialNAD(P)⁺ dependent malic enzyme (GenBank Accession No. M55905:gctgagcatcgccagggcgggcggcagggcgcggcctctccgccgggtgtacctcctgtcgcggcgcgagacctctggtgaaagaaaagatgttgtcccggttaagagtagtttccaccacttgtactttggcatgtcgacatttgcacataaaagaaaaaggcaagccacttatgctgaacccaagaacaaacaagggaatggcatttactttacaagaacgacaaatgcttggtcttcaaggacttctacctcccaaaatagagacacaagatattcaagccttacgatttcatagaaacttgaagaaaatgactagccctttggaaaaatatatctacataatgggaatacaagaaagaaatgagaaattgttttatagaatactgcaagatgacattgagagtttaatgccaattgtatatacaccgacggttggtcttgcctgctcccagtatggacacatctttagaagacctaagggattatttatttcgatctcagacagaggtcatgttagatcaattgtggataactggccagaaaatcatgttaaggctgttgtagtgactgatggagagagaattctgggtcttggagatctgggtgtctatggaatgggaattccagtaggaaaactttgtttgtatacagcttgtgcaggaatacggcctgatagatgcctgccagtgtgtattgatgtgggaactgataatatcgcactcttaaaagacccattttacatgggcttgtaccagaaacgagatcgcacacaacagtatgatgacctgattgatgagtttatgaaagctattactgacagatatggccggaacacactcattcagttcgaagactttggaaatcataatgcattcaggttcttgagaaagtaccgagaaaaatattgtactttcaatgatgatattcaagggacagctgcagtagctctagcaggtcttcttgcagcacaaaaagttattagtaaaccaatctccgaacacaaaatcttattccttggagcaggagaggctgctcttggaattgcaaatcttatagttatgtctatggtagaaaatggcctgtcagaacaagaggcacaaaagaaaatctggatgtttgacaagtatggtttattagttaagggacggaaagcaaaaatagatagttatcaggaaccatttactcactcagccccagagagcatacctgatacttttgaagatgcagtgaatatactgaagccttcaactattattggagttgcaggtgctggccgtcttttcactcctgatgtaatcagagccatggcctctatcaatgaaaggcctgtaatatttgcattaagtaatcctacagcacaggcagagtgcacggctgaagaagcatatacacttacagagggcaggtgtttgtttgccagtggcagtccatttgggccagtgaaacttacagatgggcgagtctttacaccaggtcaaggaaacaatgtttatatttttccaggtgtggctttagctgttattctctgtaacacccggcatattagtgacagtgttttcctagaagctgcaaaggccctgacaagccaattgacagatgaagagctagcccaagggagactttacccaccgcttgctaatattcaggaagtttctattaacattgctattaaagttacagaatacctatatgctaataaaatggctttccgatacccagaacctgaagacaaggccaaatatgttaaagaaagaacatggcggagtgaatatgattccctgctgccagatgtgtatgaatggccagaatctgcatcaagccctcctgtgataacagaatagaagcactcccctgataaatactttctgtgctccagggaaccccttttttcagacaagaagagataatgtcttcagtttt (SEQ ID NO: 18)),mitochondrial intermediate peptidase precursor (GenBank Accession No,U80034:gcggagcgcgcgctcccagcgaaagcagcagggcagggatctgcgttggaggaagggactgctctggtgctagaatgctgtgcgtcggaaggctgggcggcttgggagccagagcagcagctctgccgccccgccgggcgggccggggaagcctcgaagccgggatccgggcccgaagggtcagcaccagctggtctcccgtgggcgccgccttcaatgtcaagccccagggcagccgcttggacctgttcggcgagcgggcgcgtctttttggagttcctgagctgagtgccccagaaggatttcatattgcacaagaaaaagccttgagaaagacagaattgcttgtggaccgtgcatgttccaccccacctgggccccagaccgtgctgatcttcgatgagctctcggattccttatgcagagtggccgacttggctgattttgtgaaaatcgctcaccctgagccagcattcagagaagctgcggaagaagcttgtagaagtattggcaccatggtagagaagttgaacacaaatgtggatttatatcaaagtttgcaaaaattactagctgataaaaaacttgtggattcccttgatccagaaacaaggcgagtggctgaactgtttatgtttgattttgaaattagtggaatccatctagacaaacaaaagcgtaaaagagcagtggacctcaatgttaaaatcttggatttgagtagtacatttcttatgggaaccaattttcccaacaagattgagaagcatctcttaccagaacacattcgtcgtaactttacatctgctggggatcatatcataattgatggtctccacgcagaatcaccagatgacttggtgcgagaagctgcttataaaatttttctttatcccaatgctggtcaattgaaatgtttagaagaattgctcagcagcagagatcttctggcaaagttggtggggtattccacgttttctcacagggctctccaaggaacgatagctaaaaatccagagactgtcatgcagttccttgaaaaactatctgacaaactttctgaaagaactctgaaagattttgagatgatacgagggatgaaaatgaaactgaatgctcaaaattccgaagtaatgccctgggaccccccttactacagtggtgtgattcgtgcagaaaggtataatattgagcccagcctatattgcccgtttttctctcttggagcatgcatggaaggcctgaatattttgcttaacagactgttggggatttcattatatgcagagcagcctgcaaaaggagaggtgtggagcgaagatgtccgaaaactggctgttgttcatgaatctgaaggattgttggggtacatttactgtgatttttttcagcgagcagacaaaccacatcaggattgccatttcactatccgtggaggcagactaaaggaagatggagactatcaactcccacttgtagttcttatgctgaatcttccccgttcctcaaggagttctccaactttgctaactcctggcatgatggaaaatcttttccatgaaatgggacatgccatgcattcaatgctaggacgtactcgttaccaacacgtcactgggaccaggtgccctactgattttgctgaggttccttctattctgatggagtactttgcaaatgattatcgagtagttaaccaatttgccagacattatcagactggacagccactgccaaaaaatatggtgtctcgtctttgtgaatctaaaaaggtttgtgctgcagctgatatgcaacttcaggtcttttatgccactctggatcaaatctaccatgggaagcatcccctgaggaattcaaccacagacattctcaaggaaacacaagagaaattctatggcctaccatatgttccaaatactgcctggcagctgcgattcagccacctcgtggggtatggtgctagatattactcttacctcatgtccagagcggtcgcctccatggtttggaaggagtgttttctacaggatcctttcaacagggctgccggggagcgctatcgcagggagatgctggcccacggtggaggcagggagcccatgctcatggttgaaggtatgcttcagaagtgtccttctgttgatgacttcgtaagtgccctcgtttccgacttggatctggacttcgaaactttcctcatggattctgaataaaagaaacactctacacctctaatcaaggtcaact tggaataaat aatttgtttt aattaaaaaa aaaaaaaaaaaa (SEQ ID NO: 19)), andcyclin H (GenBank Accession No. U11791:ggacgctgatgcgtttgggttctcgtctgcagaccctctggacctggtcacgattccataatgtaccacaacagtagtcagaagcggcactggaccttctccagcgaggagcagctggcaagactgcgggctgacgccaaccgcaaattcagatgcaaagccgtggccaacgggaaggttcttccgaatgatccagtctttcttgagcctcatgaagaaatgacactctgcaaatactatgagaaaaggttattggaattctgttcggtgtttaagccagcaatgccaagatctgttgtgggtacggcttgtatgtatttcaaacgtttttatcttaataactcagtaatggaatatcaccccaggataataatgctcacttgtgcatttttggcctgcaaagtagatgaattcaatgtatctagtcctcagtttgttggaaacctccgggagagtcctcttggacaggagaaggcacttgaacagatactggaatatgaactacttcttatacagcaacttaatttccaccttattgtccacaatccttacagaccatttgagggcttcctcatcgacttaaagacccgctatcccatattggagaatccagagattttgaggaaaacagctgatgactttcttaatagaattgcattgacggatgcttaccttttatacacaccttcccaaattgccctgactgccattttatctagtgcctccagggctggaattactatggaaagttatttatcagagagtctgatgctgaaagagaacagaacttgcctgtcacagttactagatataatgaaaagcatgagaaacttagtaaagaagtatgaaccacccagatctgaagaagttgctgttctgaaacagaagttggagcgatgtcattctgctgagcttgcacttaacgtaatcacgaagaagaggaaaggctatgaagatgatgattacgtctcaaagaaatccaaacatgaggaggaagaatggactgatgacgacctggtagaatctctctaaccatttgaagttgatttctcaatgctaactaatcaagagaagtaggaagcatatcaaacgtttaactttatttaaaaagtataatgtgaaaacataaaatatattaaaacttttctattgttttctttccctttcacagtaactttatgtaaaataaaccatcttcaaaag(SEQ ID NO: 20)), in a tumor cell collected from a cancer patienttreated with an anticancer agent represented by the following generalformula (I):

wherein A represents benzene which may be substituted with cyano orsulfonamido, B represents benzene which may be substituted with methyl,ethyl, n-propyl, or isopropyl, C represents pyrrole which may besubstituted with cyano or a chlorine atom, W represents a single bond, Xrepresents —NH—, Y represents a carbon atom, and Z represents —NH—, and2) determining that the tumor cell is susceptible to the anticanceragent when the expression level or levels of the gene or genes decreasein comparison with those in a tumor cell collected from the cancerpatient before the treatment with the anticancer agent.
 2. A method fortesting susceptibility of a tumor cell to an anticancer agent, whichcompromises: 1) causing an anticancer agent to act on a tumor cellcollected from a cancer patient, said anticancer agent being representedby the following general formula (I):

wherein A represents benzene which may be substituted with cyano orsulfonamido, B represents benzene which may be substituted with methyl,ethyl, n-propyl, or isopropyl, C represents pyrrole which may besubstituted with cyano or a chlorine atom, W represents a single bond, Xrepresents —NH—, Y represents a carbon atom, and Z represents —NH— 2)measuring an expression level or levels of a gene or genes selected fromthe group consisting of the genes glutathione synthetase (GenBankAccession No. U34683: gggagaaccgttcgcggaggaaaggcgaactagtgttgggatggccaccaactgggggagcctcttgcaggataaacagcagctagaggagctggcacggcaggccgtggaccgggccctggctgagggagtattgctgaggacctcacaggagcccacttcctcggaggtggtgagctatgccccattcacgctcttcccctcactggtccccagtgccctgctggagcaagcctatgctgtgcagatggacttcaacctgctagtggatgctgtcagccagaacgctgccttcctggagcaaactctttccagcaccatcaaacaggatgactttaccgctcgtctctttgacatccacaagcaagtcctaaaagagggcattgcccagactgtgttcctgggcctgaatcgctcagactacatgttccagcgcagcgcagatggctccccagccctgaaacagatcgaaatcaacaccatctctgccagctttgggggcctggcctcccggaccccagctgtgcaccgacatgttctcagtgtcctgagtaagaccaaagaagctggcaagatcctctctaataatcccagcaagggactggccctgggaattgccaaagcctgggagctctacggctcacccaatgctctggtgctactgattgctcaagagaaggaaagaaacatatttgaccagcgtgccatagagaatgagctactggccaggaacatccatgtgatccgacgaacatttgaagatatctctgaaaaggggtctctggaccaagaccgaaggctgtttgtggatggccaggaaattgctgtggtttacttccgggatggctacatgcctcgtcagtacagtctacagaattgggaagcacgtctactgctggagaggtcacatgctgccaagtgcccagacattgccacccagctggctgggactaagaaggtgcagcaggagctaagcaggccgggcatgctggagatgttgctccctggccagcctgaggctgtggcccgcctccgcgccacctttgctggcctctactcactggatgtgggtgaagaaggggaccaggccatcgccgaggcccttgctgcccctagccggtttgtgctaaagccccagagagagggtggaggtaacaacctatatggggaggaaatggtacaggccctgaaacagctgaaggacagtgaggagagggcctcctacatcctcatggagaagatcgaacctgagccttttgagaattgcctgctacggcctggcagccctgcccgagtggtccagtgcatttcagagctgggcatctttggggtctatgtcaggcaggaaaagacactcgtgatgaacaagcacgtggggcatctacttcgaaccaaagccatcgagcatgcagatggtggtgtggcagcgggagtggcagtcctggacaacccataccctgtgtgagggcacaaccaggccacgggaccttctatcctctgtatttgtcattcctctcctagccctcctgaggggtatcctcctaaagacctccaaagtttttatggaagggtaaatactggtaccttcccccagctttccatctgaggaccagaaaagttgtgtctcccttagatgagatctagacgcccccaaatccttgagatgtgggtatagctcagggtaagctgctctgaggtaaaggtccatgaaccctgccccactcctgtcagcccctcatcagccttttcagcaggttccagtgcctgacttgggataggactgagtggtaggaggagggggagtggaggggcatagcctttccctaattctgccttaaataaaactgcattgctgt (SEQ ID NO: 17)), mitochondrialNAD(P)⁺dependent malic enzyme (GenBank Accession No. M55905:gctgagcatcgccagggcgggcggcagggcgcggcctctccgccgggtgtacctcctgtcgcggcgcgagacctctggtgaaagaaaagatgttgtcccggttaagagtagtttccaccacttgtactttggcatgtcgacatttgcacataaaagaaaaaggcaagccacttatgctgaacccaagaacaaacaagggaatggcatttactttacaagaacgacaaatgcttggtcttcaaggacttctacctcccaaaatagagacacaagatattcaagccttacgatttcatagaaacttgaagaaaatgactagccctttggaaaaatatatctacataatgggaatacaagaaagaaatgagaaattgttttatagaatactgcaagatgacattgagagtttaatgccaattgtatatacaccgacggttggtcttgcctgctcccagtatggacacatctttagaagacctaagggattatttatttcgatctcagacagaggtcatgttagatcaattgtggataactggccagaaaatcatgttaaggctgttgtagtgactgatggagagagaattctgggtcttggagatctgggtgtctatggaatgggaattccagtaggaaaactttgtttgtatacagcttgtgcaggaatacggcctgatagatgcctgccagtgtgtattgatgtgggaactgataatatcgcactcttaaaagacccattttacatgggcttgtaccagaaacgagatcgcacacaacagtatgatgacctgattgatgagtttatgaaagctattactgacagatatggccggaacacactcattcagttcgaagactttggaaatcataatgcattcaggttcttgagaaagtaccgagaaaaatattgtactttcaatgatgatattcaagggacagctgcagtagctctagcaggtcttcttgcagcacaaaaagttattagtaaaccaatctccgaacacaaaatcttattccttggagcaggagaggctgctcttggaattgcaaatcttatagttatgtctatggtagaaaatggcctgtcagaacaagaggcacaaaagaaaatctggatgtttgacaagtatggtttattagttaagggacggaaagcaaaaatagatagttatcaggaaccatttactcactcagccccagagagcatacctgatacttttgaagatgcagtgaatatactgaagccttcaactattattggagttgcaggtgctggccgtcttttcactcctgatgtaatcagagccatggcctctatcaatgaaaggcctgtaatatttgcattaagtaatcctacagcacaggcagagtgcacggctgaagaagcatatacacttacagagggcaggtgtttgtttgccagtggcagtccatttgggccagtgaaacttacagatgggcgagtctttacaccaggtcaaggaaacaatgtttatatttttccaggtgtggctttagctgttattctctgtaacacccggcatattagtgacagtgttttcctagaagctgcaaaggccctgacaagccaattgacagatgaagagctagcccaagggagactttacccaccgcttgctaatattcaggaagtttctattaacattgctattaaagttacagaatacctatatgctaataaaatggctttccgatacccagaacctgaagacaaggccaaatatgttaaagaaagaacatggcggagtgaatatgattccctgctgccagatgtgtatgaatggccagaatctgcatcaagccctcctgtgataacagaatagaagcactcccctgataaatactttctgtgctccagggaaccccttttttcagacaagaagagataatgtcttcagtttt (SEQ ID NO: 18)),mitochondrial intermediate peptidase precursor (GenBank Accession No,U80034:gcggagcgcgcgctcccagcgaaagcagcagggcagggatctgcgttggaggaagggactgctctggtgctagaatgctgtgcgtcggaaggctgggcggcttgggagccagagcagcagctctgccgccccgccgggcgggccggggaagcctcgaagccgggatccgggcccgaagggtcagcaccagctggtctcccgtgggcgccgccttcaatgtcaagccccagggcagccgcttggacctgttcggcgagcgggcgcgtctttttggagttcctgagctgagtgccccagaaggatttcatattgcacaagaaaaagccttgagaaagacagaattgcttgtggaccgtgcatgttccaccccacctgggccccagaccgtgctgatcttcgatgagctctcggattccttatgcagagtggccgacttggctgattttgtgaaaatcgctcaccctgagccagcattcagagaagctgcggaagaagcttgtagaagtattggcaccatggtagagaagttgaacacaaatgtggatttatatcaaagtttgcaaaaattactagctgataaaaaacttgtggattcccttgatccagaaacaaggcgagtggctgaactgtttatgtttgattttgaaattagtggaatccatctagacaaacaaaagcgtaaaagagcagtggacctcaatgttaaaatcttggatttgagtagtacatttcttatgggaaccaattttcccaacaagattgagaagcatctcttaccagaacacattcgtcgtaactttacatctgctggggatcatatcataattgatggtctccacgcagaatcaccagatgacttggtgcgagaagctgcttataaaatttttctttatcccaatgctggtcaattgaaatgtttagaagaattgctcagcagcagagatcttctggcaaagttggtggggtattccacgttttctcacagggctctccaaggaacgatagctaaaaatccagagactgtcatgcagttccttgaaaaactatctgacaaactttctgaaagaactctgaaagattttgagatgatacgagggatgaaaatgaaactgaatgctcaaaattccgaagtaatgccctgggaccccccttactacagtggtgtgattcgtgcagaaaggtataatattgagcccagcctatattgcccgtttttctctcttggagcatgcatggaaggcctgaatattttgcttaacagactgttggggatttcattatatgcagagcagcctgcaaaaggagaggtgtggagcgaagatgtccgaaaactggctgttgttcatgaatctgaaggattgttggggtacatttactgtgatttttttcagcgagcagacaaaccacatcaggattgccatttcactatccgtggaggcagactaaaggaagatggagactatcaactcccacttgtagttcttatgctgaatcttccccgttcctcaaggagttctccaactttgctaactcctggcatgatggaaaatcttttccatgaaatgggacatgccatgcattcaatgctaggacgtactcgttaccaacacgtcactgggaccaggtgccctactgattttgctgaggttccttctattctgatggagtactttgcaaatgattatcgagtagttaaccaatttgccagacattatcagactggacagccactgccaaaaaatatggtgtctcgtctttgtgaatctaaaaaggtttgtgctgcagctgatatgcaacttcaggtcttttatgccactctggatcaaatctaccatgggaagcatcccctgaggaattcaaccacagacattctcaaggaaacacaagagaaattctatggcctaccatatgttccaaatactgcctggcagctgcgattcagccacctcgtggggtatggtgctagatattactcttacctcatgtccagagcggtcgcctccatggtttggaaggagtgttttctacaggatcctttcaacagggctgccggggagcgctatcgcagggagatgctggcccacggtggaggcagggagcccatgctcatggttgaaggtatgcttcagaagtgtccttctgttgatgacttcgtaagtgccctcgtttccgacttggatctggacttcgaaactttcctcatggattctgaataaaagaaacactctacacctctaatcaaggtcaact tggaataaat aatttgtttt aattaaaaaa aaaaaaaaaaaa (SEQ ID NO: 19)), andcyclin H (GenBank Accession No. U11791:ggacgctgatgcgtttgggttctcgtctgcagaccctctggacctggtcacgattccataatgtaccacaacagtagtcagaagcggcactggaccttctccagcgaggagcagctggcaagactgcgggctgacgccaaccgcaaattcagatgcaaagccgtggccaacgggaaggttcttccgaatgatccagtctttcttgagcctcatgaagaaatgacactctgcaaatactatgagaaaaggttattggaattctgttcggtgtttaagccagcaatgccaagatctgttgtgggtacggcttgtatgtatttcaaacgtttttatcttaataactcagtaatggaatatcaccccaggataataatgctcacttgtgcatttttggcctgcaaagtagatgaattcaatgtatctagtcctcagtttgttggaaacctccgggagagtcctcttggacaggagaaggcacttgaacagatactggaatatgaactacttcttatacagcaacttaatttccaccttattgtccacaatccttacagaccatttgagggcttcctcatcgacttaaagacccgctatcccatattggagaatccagagattttgaggaaaacagctgatgactttcttaatagaattgcattgacggatgcttaccttttatacacaccttcccaaattgccctgactgccattttatctagtgcctccagggctggaattactatggaaagttatttatcagagagtctgatgctgaaagagaacagaacttgcctgtcacagttactagatataatgaaaagcatgagaaacttagtaaagaagtatgaaccacccagatctgaagaagttgctgttctgaaacagaagttggagcgatgtcattctgctgagcttgcacttaacgtaatcacgaagaagaggaaaggctatgaagatgatgattacgtctcaaagaaatccaaacatgaggaggaagaatggactgatgacgacctggtagaatctctctaaccatttgaagttgatttctcaatgctaactaatcaagagaagtaggaagcatatcaaacgtttaactttatttaaaaagtataatgtgaaaacataaaatatattaaaacttttctattgttttctttccctttcacagtaactttatgtaaaataaaccatcttcaaaag(SEQ ID NO: 20)), in the tumor cell, and 3) determining that the tumorcell is susceptible to the anticancer agent the expression level orlevels of the gene or genes decrease in comparison with those in anuntreated tumor cell.
 3. The method according to claim 1, wherein theexpression level or levels of gene or genes are measured by quantifyingan RNA or RNAs which are a transcription product or products of the geneor genes by using a DNA microarray.
 4. The method according to claim 1,wherein the expression level or levels of the gene or genes are measuredby quantifying an RNA or RNAs which are a transcription product orproducts of the gene or genes by quantitative PCR.
 5. The methodaccording to claim 2, wherein the expression level or levels of gene orgenes are measured by quantifying an RNA or RNAs which are atranscription product or products of the gene or genes by using a DNAmicroarray.
 6. The method according to claim 2, wherein the expressionlevel or levels of the gene or genes are measured by quantifying an RNAor RNAs which are a transcription product or products of the gene orgenes by quantitative PCR.
 7. The method according to claim 1, whereinsaid anticancer agent isN-(3-chloro-7-indolyl)-1,4-benzenedisulfonamide.
 8. The method accordingto claim 2, wherein said anticancer agent isN-(3-chloro-7-indolyl)-1,4-benzenesulfonamide.